Strategies for the prevention or treatment of viral infections focus mainly on two targets: the viral surface proteins or the viral replication system. Our efforts focus on the packaged replicase of nonsegmented negative strand viruses. Little is known on the molecular level about the essential, viral specific mechanisms involved in transcription and replication of these viruses. Our goal is to identify and to characterize in detail viral specific mechanisms which could be specifically inactivated without affecting the host cell. With respect to the study of the RNA dependent RNA polymerase of nonsegmented negative strand viruses such as measles, mumps, rabies, etc., vesicular stomatitis virus has become the prototype virus. The multifunctional VSV RNA polymerase functions as a transcriptase and as a replicase and aberrations of its functions can indirectly affect the cytopathogenicity of the virus. The VSV polymerase complex consists of the large viral protein L, the small NS phosphoprotein, and the genomic ribonucleocapsid template. These proteins are absolutely required for the synthesis of five viral messenger RNAs as well as the genomic RNA during replication. Complete cDNA of these genes, isolated in our lab, are being used for our studies. Using recombinant, in vitro synthesized, NS deletion mutant proteins, purified L, ribonucleocapsids, and specific antibodies, we have identified and mapped two binding domains in opposite halves of the NS protein. The data suggests that the phosphoprotein NS is a bivalent protein which binds the catalytic subunit L of the polymerase complex at its amino terminal third and the ribonucleocapsid template at the carboxy terminal half. The binding site for L contains the constitutively phosphorylated, highly negatively charged region of the NS protein while the binding site for the ribonucleocapsid involves the 21 highly conserved carboxy terminal amino acids.